Strain-specific response to anaesthetics and analgesics in rat and rabbit : A pharmacogenetic approach

The response of (laboratory) animals to anaesthetics and analgesics is known to show intraspecies variability. Apart from environmental influences, this may also be caused by genetic factors. In this thesis, rabbit and rat inbred strains were used to identify differences in response to anaesthetics and analgesics in order to provide parameters for a genetic analysis to identify candidate genes involved in this variability. The response to single dose iv bolus injections of propofol, ketamine, medetomidine and xylazine was assessed in the AX/JU and IIIVO/JU inbred rabbit strains using the time between injection and loss of righting reflex and the interval between loss and regain of righting reflex (sleep time). IIIVO/JU had a significantly longer sleep time than AX/JU when propofol and ketamine were administered. Xylazine induced severe excitation in AX/JU. Medetomidine did not induce loss of righting in all male and in six out of eight female AX/JU. The biotransformation rate of medetomidine was determined in both strains as well as the activities of the major hepatic cytochrome P450 (CYP) isoenzymes. The formation rate of hydroxymedetomidine and medetomidine carboxylic acid was significantly higher in AX/JU. Significant correlations were found between the formation rates of both metabolites and CYP2D and CYP2E like activity, as well as between these activities and the sleep time. Using amplified fragment length polymorphism markers a linkage map was constructed that was used for testing co-segregation of markers with the medetomidine metabolite formation rates in F2 intercross progeny. Significant quantitative trait loci (QTLs) were thus located on rabbit chromosomes 1 (near the albino and chloride channel locus) and 18 (near the CYP2E1 locus). When MQM (multiple-QTL model or marker-QTL-marker) mapping was used, an additional QTL (sleep time) was found on an unknown linkage group. The response of eight rat inbred strains ACI, BN, COP, F344, LEW, SHR, WAG and WKY to single doses of two analgesics (buprenorphine, nalbuphine) and three anaesthetics (propofol, medetomidine and ketamine) was determined. The analgesic response was measured using a warm water tail withdrawal procedure. The anaesthetic response was assessed in the same way as with the rabbits. ACI was hyperresponsive to buprenorphine whereas no effect was seen in F344 and WKY. None of the strains were responsive to nalbuphine. For all three anaesthetics significant strain differences in sleep time were found. F344 and BN appeared relatively resistant to medetomidine. ACI and BN appeared hypersensitive to ketamine. In all cases the sleep time of albino strains was significantly longer than pigmented strains. The behavioural response to buprenorphine was assessed in ACI and BN by the Laboratory Animal Behaviour Registration and Analysis System (LABORAS). Apart from significant strain differences in unchallenged behaviour, ACI responded to buprenorphine with a decrease in relative duration of locomotion, drinking and eating. In contrast, both strains showed increases in grooming. In conclusion this thesis describes significant rabbit and rat strain differences in response to anaesthetics and analgesics. The differences in response to medetomidine have been genetically analyzed in the rabbit and candidate genes have been identified

Comparison of 2 blood sampling methods in mice to increase animal welfare and the reliability of experimental results.

There is a strong need to gain systematic knowledge about the potential impact of routine procedures on laboratory animals to ensure animal welfare and reliability of experimental results. Blood sampling is a frequently used procedure within laboratory animal research. Two of the most common anesthesia-free techniques to obtain blood in mice are the saphenous venipuncture and submandibular bleeding. It is common practice that these methods are applied repeatedly to 1 individual. The impact of both techniques, either performed singly or repeatedly, on the animal’s welfare is not fully known yet. We directly compared the effects of both sampling methods on physiological stress responses, behavior, tissue damage, and procedure duration in 2 frequently used mouse strains: C57BL/6 and BALB/c mice (n = 45/strain). The mice were randomly assigned to 3 treatment groups (saphenous venipuncture, submandibular bleeding, control) of 15 mice/strain and were sampled weekly (80 μl/ blood sample) during 4 wk for the experimental groups and 5 wk for the control group. To evaluate the acute physiological stress response, plasma corticosterone levels were determined. As an indicator of chronic stress, body weights were recorded once a week and the thymus was weighed after necropsy. Behavior was recorded using video cameras in front of the cages and afterwards analyzed by a well-trained observer using event logging software. Tissue damage was evaluated by scoring pre-and postmortem hemorrhages and hematomas at the injection site and by measuring alanine transaminase, aspartate aminotransferase, and creatine kinase plasma levels. Submandibular bleeding appeared to be less time consuming (P<0.05) while providing higher sample volumes. However, this method led to increased acute stress responses in C57BL/6 mice (P = 0.03). All animals of both strains undergoing this procedure expressed more pain-related behavior (P < 0.05) and the extent of tissue damage at the injection site was significantly higher when compared to the saphenous venipuncture (P < 0.05). Thus, in order to avoid the undesirable effects of stress on experimental outcomes and to reduce the extent of tissue damage, saphenous venipuncture should be preferred above submandibular bleeding whenever possible

Comparison of 2 blood sampling methods in mice to increase animal welfare and the reliability of experimental results

There is a strong need to gain systematic knowledge about the potential impact of routine procedures on laboratory animals to ensure animal welfare and reliability of experimental results. Blood sampling is a frequently used procedure within laboratory animal research. Two of the most common anesthesia-free techniques to obtain blood in mice are the saphenous venipuncture and submandibular bleeding. It is common practice that these methods are applied repeatedly to 1 individual. The impact of both techniques, either performed singly or repeatedly, on the animal’s welfare is not fully known yet. We directly compared the effects of both sampling methods on physiological stress responses, behavior, tissue damage, and procedure duration in 2 frequently used mouse strains: C57BL/6 and BALB/c mice (n = 45/strain). The mice were randomly assigned to 3 treatment groups (saphenous venipuncture, submandibular bleeding, control) of 15 mice/strain and were sampled weekly (80 μl/ blood sample) during 4 wk for the experimental groups and 5 wk for the control group. To evaluate the acute physiological stress response, plasma corticosterone levels were determined. As an indicator of chronic stress, body weights were recorded once a week and the thymus was weighed after necropsy. Behavior was recorded using video cameras in front of the cages and afterwards analyzed by a well-trained observer using event logging software. Tissue damage was evaluated by scoring pre-and postmortem hemorrhages and hematomas at the injection site and by measuring alanine transaminase, aspartate aminotransferase, and creatine kinase plasma levels. Submandibular bleeding appeared to be less time consuming (P<0.05) while providing higher sample volumes. However, this method led to increased acute stress responses in C57BL/6 mice (P = 0.03). All animals of both strains undergoing this procedure expressed more pain-related behavior (P < 0.05) and the extent of tissue damage at the injection site was significantly higher when compared to the saphenous venipuncture (P < 0.05). Thus, in order to avoid the undesirable effects of stress on experimental outcomes and to reduce the extent of tissue damage, saphenous venipuncture should be preferred above submandibular bleeding whenever possible

Use of pulsed-high hydrostatic pressure treatment to decrease patulin in apple juice

This study was aimed at reducing patulin content of apple juice using a non-thermal method, namely pulsed-high hydrostatic pressure (p-HHP). Commercially available clear apple juice was contaminated artificially with different concentrations of patulin (5, 50 and 100 ppb). Then, the samples were processed 5 min at different pressure treatments (300–500 MPa) in combination with different temperatures (30–50°C) and pulses (6 pulses × 50 s and 2 pulses × 150 s). To compare the impact of pulses, single pulse of high hydrostatic pressure (HHP) treatment was also applied with the same pressure/temperature combinations and holding time. Results indicated that pressure treatment in combination with mild heat and pulses reduced the levels of patulin in clear apple juice up to 62.11%. However, reduction rates did not follow a regular pattern. p-HHP was found to be more effective in low patulin concentrations, whereas HHP was more effective for high patulin concentrations. To the best of our knowledge, this is the first study using p-HHP to investigate the reduction of patulin content in apple juice

Comparison of 2 blood sampling methods in mice to increase animal welfare and the reliability of experimental results

There is a strong need to gain systematic knowledge about the potential impact of routine procedures on laboratory animals to ensure animal welfare and reliability of experimental results. Blood sampling is a frequently used procedure within laboratory animal research. Two of the most common anesthesia-free techniques to obtain blood in mice are the saphenous venipuncture and submandibular bleeding. It is common practice that these methods are applied repeatedly to 1 individual. The impact of both techniques, either performed singly or repeatedly, on the animal’s welfare is not fully known yet. We directly compared the effects of both sampling methods on physiological stress responses, behavior, tissue damage, and procedure duration in 2 frequently used mouse strains: C57BL/6 and BALB/c mice (n = 45/strain). The mice were randomly assigned to 3 treatment groups (saphenous venipuncture, submandibular bleeding, control) of 15 mice/strain and were sampled weekly (80 μl/ blood sample) during 4 wk for the experimental groups and 5 wk for the control group. To evaluate the acute physiological stress response, plasma corticosterone levels were determined. As an indicator of chronic stress, body weights were recorded once a week and the thymus was weighed after necropsy. Behavior was recorded using video cameras in front of the cages and afterwards analyzed by a well-trained observer using event logging software. Tissue damage was evaluated by scoring pre-and postmortem hemorrhages and hematomas at the injection site and by measuring alanine transaminase, aspartate aminotransferase, and creatine kinase plasma levels. Submandibular bleeding appeared to be less time consuming (P<0.05) while providing higher sample volumes. However, this method led to increased acute stress responses in C57BL/6 mice (P = 0.03). All animals of both strains undergoing this procedure expressed more pain-related behavior (P < 0.05) and the extent of tissue damage at the injection site was significantly higher when compared to the saphenous venipuncture (P < 0.05). Thus, in order to avoid the undesirable effects of stress on experimental outcomes and to reduce the extent of tissue damage, saphenous venipuncture should be preferred above submandibular bleeding whenever possible